FIELD OF THE INVENTION

The present invention relates to a process of preparing a dry microbial formulation. The present invention particularly relates to a process of preparing a dry microbial formulation with retained stability of micro-organisms in the formulation after the drying process.

BACKGROUND

Freezing, freeze-drying and spray drying are important biological application technology, and this technology is applied in the animal feed production because of the fast development of feedstuff in recent years. One of the important steps is use of a suitable desiccation protectant in a microbial formulation for stability of micro-organisms.

Commonly used drying media for the stabilization of micro-organisms are glycerol, disaccharides, compatible solutes like trehalose, animal-source ingredients such as bovine albumin, skim milk solutions, or animal sourced proteins such as casein. Animal source cryoprotectants enable micro-organisms to survive freeze drying and, also prolongs the self-life of the micro-organisms. Due to the high demand of freeze-dried microbial formulation, there is a need for an alterntative to animal source cryoprotectant. In addition, there are restrictions from regulatory authorities of most countries to limit usage of animal source products.

There is a need to retain the microbial stability of micro-organisms during the complex freeze- drying or spray drying process without using animal source cryoprotectants.

SUMMARY OF THE CLAIMED INVENTION

The present invention provides formulations and processes for retaining or enhancing the stability of micro-organisms after the drying process.

In one aspect, a process of preparing a dry microbial formulation comprises adding one or more desiccation protectants to a fermentate of one or more micro-organisms. The one or more desiccation protectants comprises one or more non-animal source proteins. The one or more desiccation protectant enables the micro-organism to have desiccation tolerance. The one or more desiccation protectants is mixed with the fermentate of one or more micro-organisms to obtain a homogenous blend. The homogenous blend is dried using freeze drying or spray drying technique.

In another aspect, a microbial formulation comprises one or more desiccation protectants and a fermentate of one or more micro-organisms. The one or more desiccation protectants comprises one or more non-animal source proteins. The one or more desiccation protectant enables the microorganism to have desiccation tolerance. An aspect of the invention is directed to a method of protecting the cell wall of a microorganism to dessication comprising the use of non-animal source protein. In an embodiment, the method comprises the further use of a disaccharide, such as maltose.

An aspect of the invention is directed to microbial formulation comprising one or more microorganisms and a desiccation protectant comprising one or more non-animal source proteins, where said microorganism are viable after dessication such that i. at least 45% of the cells are viable after 10 weeks at 4°C; and/or ii. at least 90% of the cells are viable after 6 weeks at 4°C; and/or iii. at least 20% of the cells are viable after 4 weeks at 25°C.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the present invention may be derived by referring to the detailed description and claims when considered in connection with the Figures.

Figure 1 illustrates a flow diagram for a process of preparing a freeze-dried formulation of lactic acid bacteria.

Figure 2 shows the microbial stability of Pediococcus acidilactici and Weissella confusa over 10 weeks of storage at 4°C after freeze drying in the presence of a drying media containing either skim milk or non-animal source soytone.

Figure 3 shows the microbial stability of Weissella confusa after 6 weeks of storage at 4°Cafter spray drying in the presence of a drying media containing either skim milk, non-animal source soy protein, or non-animal source soytone.

DEFINITIONS

The disclosed embodiments relate to formulations and processes for retaining the stability of micro-organisms in dried microbial formulations.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. For the sake of brevity and/or clarity, well-known functions or constructions may not be described in detail.

As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Throughout this disclosure, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

The term "consisting of" means including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. The term "consisting essentially of' means including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

As used herein, the term “desiccation tolerance” is characterized as the ability for a strain to survive and remain stable in storage after the process of desiccation through technologies such as spray drying, freeze-drying, etc. The desiccation tolerance is usually viewed as a comparative measure, pitting one formulation or strain against another. All strains have an inate level of desiccation tolerance that varies based on physiology and natural mechanisms to survive the stressful desiccation process. For some strains, such as Bacillus spores, survival of less than 90% through a drying process is considered poor performance while in other strain, such a Gram (-) organisms, survival of 1-10% is considered a success.

As used herein, ‘stability of micro-organisms” means maintaining and/or increasing the viability, survivability, and/or CFU of one or more micro-organisms as compared to one or more controls (e.g., a control composition that is identical to a desiccation protectant composition of the present disclosure except that the control composition lacks one or more of the components found in the desiccation composition of the present disclosure). As used herein, ‘stability of micro-organisms” is further intended to mean preventing and/or decreasing the amount of death and/or rate of death of one or more micro-organisms.

As used herein, "enhanced stability" refers to an improvement in one or more characteristics of stability of micro-organisms as compared to one or more controls (e.g., a control composition that is identical to a desiccation protectant composition of the present disclosure except that the control composition lacks one or more of the components found in the desiccation composition of the present disclosure). Exemplary characteristics of stability of micro-organisms include, but are not limited to, the ability of the micro-organism population to propagate, grow, and/or be cultured after being stored for a defined period of time. A microorganism that exhibits improvement in one or more microbial stability characteristics as compared to a control microorganism when each is subjected to the same conditions (e.g., storage conditions) displays enhanced stability and can be referred to as a "stable microorganism." A desiccation protectant composition that improves one or more microbial stability characteristics of the microorganism(s) contained therein as compared to a control composition (e.g., a control composition that is identical to the desiccation protectant composition except that the control composition lacks one or more of the components found in the inoculant composition) provides enhanced stability.

As used herein, "enhanced survival" refers to an improvement in the survival rate of one or more microorganisms through processing and more specifically desiccation when using an desiccation protectant composition as compared to one or more microorganisms in a control composition (e.g., a control composition that is identical to an desiccation protectant composition of the present disclosure except that the control composition lacks one or more of the components found in the desiccation protectant composition of the present disclosure). A desiccation protectant composition that improves the survival rate of one or more of the microorganisms contained therein as compared to a control composition (e.g., a control composition that is identical to the desiccation protectant composition except that the control composition lacks one or more of the components found in the desiccation protectant composition) provides enhanced survival.

As used herein, “desiccation protectants” means elements protecting against the harmful effect of drying techniques such as the ones submitted for example in freeze-drying, freezing processes, or spray drying. In addition, desiccation protectants confer some stability through the drying process. The action of desiccation protectant will reduce loss of activity or viability during the manufacturing process and subsequently, its action improves the activity/viability of the microorganisms during storage.

As used herein, “cryoprotectants” means elements protecting against the harmful effects of low or freezing temperatures, such as the ones submitted for example in freeze-drying or freezing processes. In addition, in the case of freeze-drying or drying, it confers to the dried elements some stability through the drying process. The action of the cryoprotectant will reduce loss of activity or viability during the manufacturing process and subsequently, its action improves the activity/viability of the micro-organisms during storage.

As used herein, “fermentate” means a starting material for the present invention which is obtained by fermentation of one or more micro-organisms.

As used herein, “homogenous blend” means a composition having substantially one morphological phase in the same state.

As used herein, “freeze-drying” means lyophilisation, lyophilization, or cryodesiccation, which is used in its regular meaning as the cooling of a sample, resulting in the conversion of freeze-able solution into ice, crystallization of crystallisable solutes and the formation of an amorphous matrix comprising non-crystallizing solutes associated with unfrozen mixture, followed by evaporation (sublimation) of water from amorphous matrix. In this process the evaporation (sublimation) of the frozen water in the material is usually carried out under reducing the surrounding pressure to allow the frozen water in the material to sublimate directly from the solid phase to the gas phase. Freeze- drying typically includes the steps of pretreatment, freezing, primary drying and secondary drying. The great advantage of freeze drying is to stabilize the materials for storage.

As used herein, "spray-dried" microbes within the meaning of the invention denotes that the microbial cells are dried using a spray drying or atomizing method (synonymous), wherein a suspension of microbial cells is dispersed into fine mist-like droplets, for example, and a powder can be obtained.

As used herein, “spray drying” is a drying method where a solution or suspension containing microbial cells is sprayed into a hot drying medium, whereby the microbial cells are dried. The mixture to be sprayed can be present in the form of a solution, an emulsion, a suspension or dispersion. The mixture is atomized into millions of individual droplets with the aid of a nozzle or a spraying wheel, drastically increasing the surface. The solvent, such as water, is immediately evaporated by the hot air and is discharged. Moreover, the microbial cells are spray-dried alone. The spray drying or atomization method can be distinguished from other drying methods since the use of a nozzle or similarly acting means is required, such as a unary nozzle, hollow cone nozzle, pressure nozzle, binary nozzle externally mixing, pneumatic nozzle, binary nozzle internally mixing, atomizing disk or ultrasonic atomizer. Spray drying methods are described in the prior art and are familiar to the person skilled in the art (see Gardiner et al., Teixeira et al. (supra) or EP74050 and EP285682). Devices are known and described as relevant, such as the mini spray dryer B-191 or B-290 by Buechi Labortechnik AG (Germany) or SD-6.3-R by GEA Niro (Denmark). It is further known that arbitrary adjuvants and additives can be used.

As used herein, “non-animal source protein” means protein that are not obtained or derived from animals. Non-animal proteins can be obtained or derived from non-animal sources such as plants, microbes, etc.

While certain aspects of the present disclosure will hereinafter be described with reference to embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect the invention relates to a process of preparing a dry microbial formulation comprising adding one or more desiccation protectants to a fermentate of one or more microorganisms, mixing the one or more desiccation protectants with the fermentate to obtain a blend, thereafter drying the blend. The one or more desiccation protectants comprises a non-animal source protein. The one or more desiccation protectant enables the micro-organism to have desiccation tolerance. The blend is typically homogenous. An aspect of the invention is directed to a microbial formulation comprising at least one desiccation protectant and a fermentate of at least one microorganism, wherein the at least one desiccation protectant comprises a non-animal source protein and wherein the at least one desiccation protectant enables the micro-organism to have desiccation tolerance. An aspect of the invention is directed to a method of protecting the cell wall of a microorganism to dessication/drying/ comprising the use of non-animal source protein (as cryoprotectant). An aspect of the invention is directed to microbial formulation comprising one or more microorganisms and a desiccation protectant comprising one or more non-animal source proteins, where said microorganism are viable after dessication such that i. at least 45% of the cells are viable after 10 weeks at 4°C, such as under Dessication Method 1 ; and/or ii. at least 90% of the cells are viable after 6 weeks at 4°C, such as under Dessication Method 2; and/or iii. at least 20% of the cells are viable after 4 weeks at 25°C, such as under Dessication Method 3.

In the process of preparing a dry microbial formulation, in order to retain the properties of microbial stability of micro-organisms, the one or more desiccation protectants are added to the fermentate before the step of drying. According to the present invention, non-animal source desiccation protectant can replace the animal source desiccation protectant such as skim milk, casein, etc. which is currently widely used. The present invention eliminates the need to use desiccation protectants which are sourced from animals, milk or insects. Accordingly, an alternative aspect of the invention is directed to a process of preparing a dry microbial formulation comprising adding one or more desiccation protectants to a fermentate of one or more micro-organisms, mixing the one or more desiccation protectants with the fermentate to obtain a blend, thereafter drying the blend, wherein the dessicant protectant is free or substantially free from animal, milk or insect products. The blend is typically homogenous. Otherwise stated, an aspect relates to process of preparing a dry microbial formulation comprises adding one or more desiccation protectants to a fermentate of one or more micro-organisms, wherein the one or more desiccation protectants comprises one or more non- animal source proteins. In an embodiment of the process, the desiccation protectant is a cryoprotectant.

In an embodiment of the process, the cryoprotectant is a liquid cryoprotectant.

In an embodiment of the process, the one or more desiccation protectants enable the one or more micro-organisms to maintain the properties of microbial stability. The properties of microbial stability include survivability and shelf life of the one or more micro-organisms.

The inventor further surprisingly found that a desiccation protectant formulation comprised of non-animal source protein, a sugar, and water added to a micro-organism resulted in a microorganism with equal or enhanced desiccation tolerance when compared to the same micro-organism formulated with a desiccation protectant comprised of animal-source proteins such as skim milk as measured by survivability and stability.

In a preferred embodiment of the process, the liquid cryoprotectant comprises a non-animal source protein, a sugar, and water. In a preferred embodiment of the process, the liquid cryoprotectant includes soytone, maltose and water. In a specific embodiment of the process, the liquid cryoprotectant includes 1.0-30.0% of soytone, 0.1%-30.0% of maltose, and water. In another embodiment, the ratio of soytone to maltose to water is 1 :0.1 :99 to 3:3:4. The soytone is a peptone that is protein hydrolysates formed by enzymatic or acidic digestion of soybean. The peptone is a soluble protein formed in the early stage of protein breakdown during digestion of plant raw materials.

The one or more desiccation protectants can include a drying media for the one or more microorganisms. In a specific embodiment, a drying media for lactic acid bacteria comprises glycerol, disaccharides, compatible solutes and non-animal source protein.

In an embodiment, the non-animal source protein is a plant source protein. In another embodiment of the process, the non-animal source protein is a microbial source protein. The non- animal source protein can be protein concentrates, protein isolates, or peptones. The peptones can be obtained by acid hydrolysis or enzyme digestion.

In an embodiment, the plant source protein is a protein from cereal, pseudo cereal, grain, legume, pulse, vegetable, root, nuts, hemp, chia seeds, and/or flax seeds. The cereals, pseudo cereal, and grains can include wheat, rice, corn, amaranth, quinoa, and others which can provide protein concentrates, protein isolates, or peptones that could be used for microbial stabilization. The legumes and pulses can include soy, chickpeas, beans, peas, lentils, peanut, and others which can provide protein concentrates, protein isolates, or peptones that could be used for microbial stabilization. In a preferred embodiment, the legume is soybean. The vegetable and roots can include potato, cassava, arrowroot, and others which can provide protein concentrates, protein isolates, or peptones that could be used for microbial stabilization.

In an embodiment, the microbial source protein is a protein from yeast, yeast extract, single cell protein, and/or mycoproteins.

In a preferred embodiment, the non-animal source protein is a peptone. In a specific embodiment, the peptone is soy peptone. The peptone is a protein hydrolysate formed by enzymatic or acidic digestion of various plant based raw materials. In a preferred embodiment of the process, the sugar can be selected from the group consisting of maltodextrins, monosaccharides, disaccharides, oligosaccharides, and malt extracts. In an embodiment, the sugar may comprise any suitable monosaccharide(s) including allose, altrose, arabinose, fructose, galactose, glucose, gulose, iodose, lyxose, manose, ribose, talose, threose and/or xylose. In another embodiment, the sugar may comprise any suitable disaccharide(s) including cellobiose, chitobiose, gentiobiose, gentiobiulose, isomaltose, kojibiose, lactose, lactulose, laminaribiose, maltose (e.g. maltose monohydrate, anhydrous maltose), maltulose, mannobiose, melibiose, melibiulose, nigerose, palatinose, rutinose, rutinulose, sophorose, sucrose, trehalose, turanose, and/or xylobiose.

In a specific embodiment, the sugar is maltose. The inventor further surprisingly found that use of sugar with non-animal source protein, specifically maltose, stabilizes the cell membrane of the micro-organisms which in turns helps to retain the properties of microbial stability of the microorganism during freeze-drying.

In another embodiment, the sugar may comprise any suitable oligosaccharide(s) including fructo-oligosaccharides, galacto-oligosaccharides, mannon-oligosaccharides and/or raffinose.

In a preferred embodiment, the one or more micro-organisms comprises one or more lactic acid bacteria strains. In another preferred embodiment, the one or more lactic acid bacteria is selected from a group of consisting of Pediococcus pentosaceus, Pediococcus acidilactici, Lactobacillus salivarius, Weissella confusa, Lactobacillus parafarraginis, Lactobacillus plantarum, and Lactobacillus reuteri. In another preferred embodiment, the one or more lactic acid bacteria is selected from a group of consisting of Pediococcus pentosaceus, Pediococcus acidilactici, Lactobacillus salivarius, Weissella confusa, Lactobacillus parafarraginis, Lactobacillus plantarum, and Lactobacillus reuteri, and are viable after dessication such that i. at least 45% of the cells are viable after 10 weeks at 4°C; and/or ii. at least 90% of the cells are viable after 6 weeks at 4°C; and/or iii. at least 20% of the cells are viable after 4 weeks at 25°C.

In a specific embodiment, the one or more micro-organisms comprises one or more lactic acid bacteria selected from the group consisting of i) Pediococcus acidilactici FM18 deposited as NRRL Deposit Number B-50964, ii) Pediococcus acidilactici TY036 deposited as NRRL Deposit Number B-50959, iii) Enterococcus faecium MFF109 deposited as NRRL Deposit Number B-50960, and iv) a strain having all of the identifying characteristics of Pediococcus acidilactici FM18 deposited as NRRL Deposit Number B-50964, Pediococcus acidilactici TY036 deposited as NRRL Deposit Number B-50959, or Enterococcus faecium MFF109 deposited as NRRL Deposit Number B- 50960 or a mutant thereof.

In a specific embodiment, the one or more micro-organisms comprises one or more lactic acid bacteria selected from the group consisting of i) Pediococcus acidilactici 64-C1 deposited as NRRL B-50962, ii) Lactobacillus salivarius 64-C2 deposited as NRRL B-67320, iii) Lactobacillus salivarius 39B1 deposited as NRRL B-67178, iii) Pediococcus pentosaceus 39B2 deposited as NRRL B-67179, iv) Lactobacillus salivarius 59B deposited as NRRL B-67177, v) Lactobacillus salivarius 34B deposited as NRRL B-67180, and vi) a strain having all of the identifying characteristics of Pediococcus acidilactici 64-C1 deposited as NRRL B-50962, Lactobacillus salivarius 64-C2 deposited as NRRL B-67320, Lactobacillus salivarius 39B1 deposited as NRRL B-67178, Pediococcus pentosaceus 39B2 deposited as NRRL B-67179, Lactobacillus salivarius 59B deposited as NRRL B-67177, or Lactobacillus salivarius 34B deposited as NRRL B-67180 or a mutant thereof.

The process and formulation according to the present invention also relates to an embodiment wherein the one or more micro-organisms are those micro-organisms that are used in animal feed compositions or animal feed additive.

Non-limiting examples of a micro-organisms may be: Lactobacillus delbrueckii, Lactobacillus acetotolerans, Lactobacillus achengensis, Lactobacillus acidifarinae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus allii, Lactobacillus alvi, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylotrophicus, Lactobacillus amylovorus, Lactobacillus angrenensis, Lactobacillus animalis, Lactobacillus antri, Lactobacillus apinorum, Lactobacillus apis, Lactobacillus apodemi, Lactobacillus aquaticus, Lactobacillus argentoratensis, Lactobacillus arizonensis, Lactobacillus aviarius, Lactobacillus backii, Lactobacillus baiquanensis, Lactobacillus bambusae, Lactobacillus baoqingensis, Lactobacillus bavaricus, Lactobacillus bayanensis, Lactobacillus bifermentans, Lactobacillus binensis, Lactobacillus bobalius, Lactobacillus bombi, Lactobacillus bombicola, Lactobacillus bombintestini, Lactobacillus brantae, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus bulgaricus, Lactobacillus cacaonum, Lactobacillus camelliae, Lactobacillus capillatus, Lactobacillus carnis, Lactobacillus casei, Lactobacillus catenaformis, Lactobacillus caucasicus, Lactobacillus caviae, Lactobacillus cellobiosus, Lactobacillus cerevisiae, Lactobacillus ceti, Lactobacillus chiayiensis, Lactobacillus coleohominis, Lactobacillus colini, Lactobacillus collinoides, Lactobacillus composti, Lactobacillus concavus, Lactobacillus confusus, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curieae, Lactobacillus curtus, Lactobacillus curvatus, Lactobacillus cypricasei, Lactobacillus daoliensis, Lactobacillus daowaiensis, Lactobacillus daqingensis, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus divergens, Lactobacillus dongliensis, Lactobacillus durianis, Lactobacillus enshiensis, Lactobacillus equi, Lactobacillus equicursoris, Lactobacillus equigenerosi, Lactobacillus fabifermentans, Lactobacillus faecis, Lactobacillus farciminis, Lactobacillus farraginis, Lactobacillus ferintoshensis, Lactobacillus fermentum, Lactobacillus floricola, Lactobacillus florum, Lactobacillus formosensis, Lactobacillus fornicalis, Lactobacillus fructivorans, Lactobacillus fructosus, Lactobacillus frumenti, Lactobacillus fuchuensis, Lactobacillus fujinensis, Lactobacillus furfuricola, Lactobacillus futsaii, Lactobacillus fuyuanensis, Lactobacillus gallinarum, Lactobacillus gannanensis, Lactobacillus garii, Lactobacillus gasseri, Lactobacillus gastricus, Lactobacillus ghanensis, Lactobacillus gigeriorum, Lactobacillus ginsenosidimutans, Lactobacillus gorillae, Lactobacillus graminis, Lactobacillus halodurans, Lactobacillus halotolerans, Lactobacillus hammesii, Lactobacillus hamsteri, Lactobacillus harbinensis, Lactobacillus hayakitensis, Lactobacillus hegangensis, Lactobacillus heilongjiangensis, Lactobacillus helsingborgensis, Lactobacillus helveticus, Lactobacillus herbarum, Lactobacillus heterohiochii, Lactobacillus hilgardii, Lactobacillus hokkaidonensis, Lactobacillus hominis, Lactobacillus homohiochii, Lactobacillus hordei, Lactobacillus huachuanensis, Lactobacillus huananensis, Lactobacillus hulanensis, Lactobacillus hulinensis, Lactobacillus iners, Lactobacillus ingluviei, Lactobacillus insicii, Lactobacillus intestinalis, Lactobacillus iwatensis, Lactobacillus ixorae, Lactobacillus jensenii, Lactobacillus jiayinensis, Lactobacillus jidongensis, Lactobacillus jinshani, Lactobacillus jixianensis, Lactobacillus johnsonii, Lactobacillus kaifaensis, Lactobacillus kalixensis, Lactobacillus kandleri, Lactobacillus kedongensis, Lactobacillus kefir, Lactobacillus kefiranofaciens, Lactobacillus kefirgranum, Lactobacillus keshanensis, Lactobacillus kimbladii, Lactobacillus kimchicus, Lactobacillus kimchiensis, Lactobacillus kimchii, Lactobacillus kisonensis, Lactobacillus kitasatonis, Lactobacillus koreensis, Lactobacillus kosoi, Lactobacillus kullabergensis, Lactobacillus kunkeei, Lactobacillus lactis, Lactobacillus leichmannii, Lactobacillus lindianensis, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus maltaromicus, Lactobacillus manihotivorans, Lactobacillus mellifer, Lactobacillus mellis, Lactobacillus melliventris, Lactobacillus metriopterae, Lactobacillus micheneri, Lactobacillus mindensis, Lactobacillus minor, Lactobacillus minutus, Lactobacillus mishanensis, Lactobacillus mixtipabuli, Lactobacillus modestisalitolerans, Lactobacillus mucosae, Lactobacillus mudanjiangensis, Lactobacillus mulanensis, Lactobacillus mulengensis, Lactobacillus mulieris, Lactobacillus murinus, Lactobacillus musae, Lactobacillus nagelii, Lactobacillus namurensis, Lactobacillus nangangensis, Lactobacillus nantensis, Lactobacillus nasuensis, Lactobacillus nenjiangensis, Lactobacillus nodensis, Lactobacillus nuruki, Lactobacillus odoratitofui, Lactobacillus oeni, Lactobacillus oligo, Lactobacillus oris, Lactobacillus oryzae, Lactobacillus otakiensis, Lactobacillus ozensis, Lactobacillus panis, Lactobacillus panisapium, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus parabuchneri, Lactobacillus paracasei, Lactobacillus paracollinoides, Lactobacillus parafarraginis, Lactobacillus paragasseri, Lactobacillus parakefiri, Lactobacillus paralimentarius, Lactobacillus paraplantarum, Lactobacillus pasteurii, Lactobacillus paucivorans, Lactobacillus pentosiphilus, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus pingfangensis, Lactobacillus piscicola, Lactobacillus plajomi, Lactobacillus plantarum, Lactobacillus pobuzihii, Lactobacillus pontis, Lactobacillus porci, Lactobacillus porcinae, Lactobacillus psittaci, Lactobacillus quenuiae, Lactobacillus raoultii, Lactobacillus rapi, Lactobacillus rennini, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rimae, Lactobacillus rodentium, Lactobacillus rogosae, Lactobacillus rossiae, Lactobacillus ruminis, Lactobacillus saerimneri, Lactobacillus sakei, Lactobacillus salitolerans, Lactobacillus salivarius, Lactobacillus salsicarnum, Lactobacillus sanfranciscensis, Lactobacillus saniviri, Lactobacillus satsumensis, Lactobacillus secaliphilus, Lactobacillus selangorensis, Lactobacillus senioris, Lactobacillus senmaizukei, Lactobacillus sharpeae, Lactobacillus shenzhenensis, Lactobacillus sicerae, Lactobacillus silagei, Lactobacillus silagincola, Lactobacillus siliginis, Lactobacillus similis, Lactobacillus sobrius, Lactobacillus songbeiensis, Lactobacillus songhuajiangensis, Lactobacillus spicheri, Lactobacillus suantsaicola, Lactobacillus suantsaii, Lactobacillus suantsaiihabitans, Lactobacillus sucicola, Lactobacillus suebicus, Lactobacillus suibinensis, Lactobacillus sunkii, Lactobacillus suntoryeus, Lactobacillus taiwanensis, Lactobacillus tangyuanensis, Lactobacillus terrae, Lactobacillus thailandensis, Lactobacillus thermotolerans, Lactobacillus timberlakei, Lactobacillus timonensis, Lactobacillus tongjiangensis, Lactobacillus trichodes, Lactobacillus tucceti, Lactobacillus uli, Lactobacillus ultunensis, Lactobacillus uvarum, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus versmoldensis, Lactobacillus vespulae, Lactobacillus vini, Lactobacillus viridescens, Lactobacillus vitulinus, Lactobacillus wasatchensis, Lactobacillus wuchangensis, Lactobacillus xiangfangensis, Lactobacillus xujianguonis, Lactobacillus xylosus, Lactobacillus yamanashiensis, Lactobacillus yichunensis, Lactobacillus yilanensis, Lactobacillus yonginensis, Lactobacillus zeae, Lactobacillus zhachilii, Lactobacillus zhaodongensis, Lactobacillus zhaoyuanensis, Lactobacillus zhongbaensis, Lactobacillus zymae, Lactobacillus sp.

In an embodiment of the process, the desiccation protectant is mixed with microorganisms until evenly dispersed to obtain a homogenous blend.

In an embodiment of the process, drying of the homogenous blend is one of freezing, freeze- drying or spray-drying or combination thereof.

In a preferred embodiment of the process, the homogenous blend is frozen before freeze- drying. The homogenous blend can be frozen using various known freezing techniques such as snap freezing by pouring liquid nitrogen on the homogenous blend or storing the homogenous blend at - 80°C until frozen. In another embodiment, the homogenous blend can be frozen at a temperature of -80°C to -18°C for 15 minutes to 24 hours. The homogenous blend is retained in the frozen state until used for drying process.

In an embodiment of the process, freeze-drying is carried out for 24 hours or until the homogenous blend is dried. The time for freeze drying varies depending on the components in the formulation. In an embodiment, the homogenous blend is spray dried using any spray dryer known in the art of drying microbial formulation.

The present invention further relates to the use or the application of the dry microbial formulation according to the invention in an animal feed composition and an animal feed additive.

In a preferred aspect, the invention relates to a microbial formulation comprising one or more desiccation protectants and a fermentate of one or more micro-organisms, wherein the one or more desiccation protectants is a non-animal source protein. The one or more desiccation protectant enables the micro-organism to have desiccation tolerance. The micro-organisms in the microbial formulation has higher survival rate after drying compared to the same micro-organisms formulated with a desiccation protectant comprised of an animal source protein.

In a preferred embodiment of the microbial formulation, the one or more desiccation protectants are cryoprotectants. In an embodiment, the cryoprotectant is a liquid cryoprotectant.

In an embodiment of the microbial formulation, the one or more desiccation protectants enable the one or more micro-organisms to maintain the properties of microbial stability.

In an embodiment of the microbial formulation, the non-animal source protein is a plant source protein. In another embodiment of the microbial formulation, the non-animal source protein is a microbial source protein.

In an embodiment of the microbial formulation, the plant souce protein is a protein from cereal, pseudo cereal, grain, legume, pulse, vegetable, root, nuts, hemp, chia seeds, and/or flax seeds.

In an embodiment of the microbial formulation, the microbial source protein is a protein from yeast, yeast extract, single cell protein, and/or mycoproteins.

In a preferred embodiment of the microbial formulation, the non-animal source protein is a peptone. In a specific embodiment, the peptone is soy peptone.

In a preferred embodiment of the microbial formulation, the one or more micro-organisms comprises one or more lactic acid bacteria strains. In another preferred embodiment, the one or more lactic acid bacteria is selected from a group of consisting of Pediococcus pentosaceus, Pediococcus acidilactici or Lactobacillus salivarius, Weissella confusa, Lactobacillus parafarraginis, Lactobacillus plantarum, and Lactobacillus reuteri. In another preferred embodiment, the one or more lactic acid bacteria is selected from a group of consisting of Pediococcus pentosaceus, Pediococcus acidilactici, Lactobacillus salivarius, Weissella confusa, Lactobacillus parafarraginis, Lactobacillus plantarum, and Lactobacillus reuteri, and are viable after dessication such that i. at least 45% of the cells are viable after 10 weeks at 4°C; and/or ii. at least 90% of the cells are viable after 6 weeks at 4°C; and/or iii. at least 20% of the cells are viable after 4 weeks at 25°C. In a specific embodiment of the microbial formulation, the one or more micro-organisms comprises one or more lactic acid bacteria selected from the group consisting of i) Pediococcus acidilactici FM18 deposited as NRRL Deposit Number B-50964, ii) Pediococcus acidilactici TY036 deposited as NRRL Deposit Number B-50959, iii) Enterococcus faecium MFF109 deposited as NRRL Deposit Number B-50960, and iv) a strain having all of the identifying characteristics of Pediococcus acidilactici FM18 deposited as NRRL Deposit Number B-50964, Pediococcus acidilactici TY03Q deposited as NRRL Deposit Number B-50959, or Enterococcus faecium MFF109 deposited as NRRL Deposit Number B- 50960 or a mutant thereof.

In a specific embodiment of the microbial formulation, the one or more micro-organisms comprises one or more lactic acid bacteria selected from the group consisting of i) Pediococcus acidilactici 64-C1 deposited as NRRL B-50962, ii) Lactobacillus salivarius 64-C2 deposited as NRRL B-67320, iii) Lactobacillus salivarius 39B1 deposited as NRRL B-67178, iii) Pediococcus pentosaceus 39B2 deposited as NRRL B-67179, iv) Lactobacillus salivarius 59B deposited as NRRL B-67177, v) Lactobacillus salivarius 34B deposited as NRRL B-67180, and vi) a strain having all of the identifying characteristics of Pediococcus acidilactici 64-C1 deposited as NRRL B-50962, Lactobacillus salivarius 64-C2 deposited as NRRL B-67320, Lactobacillus salivarius 39B1 deposited as NRRL B-67178, Pediococcus pentosaceus 39B2 deposited as NRRL B-67179, Lactobacillus salivarius 59B deposited as NRRL B-67177, or Lactobacillus salivarius 34B deposited as NRRL B-67180 or a mutant thereof.

Non-limiting examples of a micro-organisms may be:

Lactobacillus delbrueckii, Lactobacillus acetotolerans, Lactobacillus achengensis, Lactobacillus acidifarinae, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus allii, Lactobacillus alvi, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylotrophicus, Lactobacillus amylovorus, Lactobacillus angrenensis, Lactobacillus animalis, Lactobacillus antri, Lactobacillus apinorum, Lactobacillus apis, Lactobacillus apodemi, Lactobacillus aquaticus, Lactobacillus argentoratensis, Lactobacillus arizonensis, Lactobacillus aviarius, Lactobacillus backii, Lactobacillus baiquanensis, Lactobacillus bambusae, Lactobacillus baoqingensis, Lactobacillus bavaricus, Lactobacillus bayanensis, Lactobacillus bifermentans, Lactobacillus binensis, Lactobacillus bobalius, Lactobacillus bombi, Lactobacillus bombicola, Lactobacillus bombintestini, Lactobacillus brantae, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus bulgaricus, Lactobacillus cacaonum, Lactobacillus camelliae, Lactobacillus capillatus, Lactobacillus carnis, Lactobacillus casei, Lactobacillus catenaformis, Lactobacillus caucasicus, Lactobacillus caviae, Lactobacillus cellobiosus, Lactobacillus cerevisiae, Lactobacillus ceti, Lactobacillus chiayiensis, Lactobacillus coleohominis, Lactobacillus colini, Lactobacillus collinoides, Lactobacillus composti, Lactobacillus concavus, Lactobacillus confusus, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curieae, Lactobacillus curtus, Lactobacillus curvatus, Lactobacillus cypricasei, Lactobacillus daoliensis, Lactobacillus daowaiensis, Lactobacillus daqingensis, Lactobacillus dextrinicus, Lactobacillus diolivorans, Lactobacillus divergens, Lactobacillus dongliensis, Lactobacillus durianis, Lactobacillus enshiensis, Lactobacillus equi, Lactobacillus equicursoris, Lactobacillus equigenerosi, Lactobacillus fabifermentans, Lactobacillus faecis, Lactobacillus farciminis, Lactobacillus farraginis, Lactobacillus ferintoshensis, Lactobacillus fermentum, Lactobacillus floricola, Lactobacillus florum, Lactobacillus formosensis, Lactobacillus fornicalis, Lactobacillus fructivorans, Lactobacillus fructosus, Lactobacillus frumenti, Lactobacillus fuchuensis, Lactobacillus fujinensis, Lactobacillus furfuricola, Lactobacillus futsaii, Lactobacillus fuyuanensis, Lactobacillus gallinarum, Lactobacillus gannanensis, Lactobacillus garii, Lactobacillus gasseri, Lactobacillus gastricus, Lactobacillus ghanensis, Lactobacillus gigeriorum, Lactobacillus ginsenosidimutans, Lactobacillus gorillae, Lactobacillus graminis, Lactobacillus halodurans, Lactobacillus halotolerans, Lactobacillus hammesii, Lactobacillus hamsteri, Lactobacillus harbinensis, Lactobacillus hayakitensis, Lactobacillus hegangensis, Lactobacillus heilongjiangensis, Lactobacillus helsingborgensis, Lactobacillus helveticus, Lactobacillus herbarum, Lactobacillus heterohiochii, Lactobacillus hilgardii, Lactobacillus hokkaidonensis, Lactobacillus hominis, Lactobacillus homohiochii, Lactobacillus hordei, Lactobacillus huachuanensis, Lactobacillus huananensis, Lactobacillus hulanensis, Lactobacillus hulinensis, Lactobacillus iners, Lactobacillus ingluviei, Lactobacillus insicii, Lactobacillus intestinalis, Lactobacillus iwatensis, Lactobacillus ixorae, Lactobacillus jensenii, Lactobacillus jiayinensis, Lactobacillus jidongensis, Lactobacillus jinshani, Lactobacillus jixianensis, Lactobacillus johnsonii, Lactobacillus kaifaensis, Lactobacillus kalixensis, Lactobacillus kandleri, Lactobacillus kedongensis, Lactobacillus kefir, Lactobacillus kefiranofaciens, Lactobacillus kefirgranum, Lactobacillus keshanensis, Lactobacillus kimbladii, Lactobacillus kimchicus, Lactobacillus kimchiensis, Lactobacillus kimchii, Lactobacillus kisonensis, Lactobacillus kitasatonis, Lactobacillus koreensis, Lactobacillus kosoi, Lactobacillus kullabergensis, Lactobacillus kunkeei, Lactobacillus lactis, Lactobacillus leichmannii, Lactobacillus lindianensis, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus maltaromicus, Lactobacillus manihotivorans, Lactobacillus mellifer, Lactobacillus mellis, Lactobacillus melliventris, Lactobacillus metriopterae, Lactobacillus micheneri, Lactobacillus mindensis, Lactobacillus minor, Lactobacillus minutus, Lactobacillus mishanensis, Lactobacillus mixtipabuli, Lactobacillus modestisalitolerans, Lactobacillus mucosae, Lactobacillus mudanjiangensis, Lactobacillus mulanensis, Lactobacillus mulengensis, Lactobacillus mulieris, Lactobacillus murinus, Lactobacillus musae, Lactobacillus nagelii, Lactobacillus namurensis, Lactobacillus nangangensis, Lactobacillus nantensis, Lactobacillus nasuensis, Lactobacillus nenjiangensis, Lactobacillus nodensis, Lactobacillus nuruki, Lactobacillus odoratitofui, Lactobacillus oeni, Lactobacillus oligo, Lactobacillus oris, Lactobacillus oryzae, Lactobacillus otakiensis, Lactobacillus ozensis, Lactobacillus panis, Lactobacillus panisapium, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus parabuchneri, Lactobacillus paracasei, Lactobacillus paracollinoides, Lactobacillus parafarraginis, Lactobacillus paragasseri, Lactobacillus parakefiri, Lactobacillus paralimentarius, Lactobacillus paraplantarum, Lactobacillus pasteurii, Lactobacillus paucivorans, Lactobacillus pentosiphilus, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus pingfangensis, Lactobacillus piscicola, Lactobacillus plajomi, Lactobacillus plantarum, Lactobacillus pobuzihii, Lactobacillus pontis, Lactobacillus porci, Lactobacillus porcinae, Lactobacillus psittaci, Lactobacillus quenuiae, Lactobacillus raoultii, Lactobacillus rapi, Lactobacillus rennini, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rimae, Lactobacillus rodentium, Lactobacillus rogosae, Lactobacillus rossiae, Lactobacillus ruminis, Lactobacillus saerimneri, Lactobacillus sakei, Lactobacillus salitolerans, Lactobacillus salivarius, Lactobacillus salsicarnum, Lactobacillus sanfranciscensis, Lactobacillus saniviri, Lactobacillus satsumensis, Lactobacillus secaliphilus, Lactobacillus selangorensis, Lactobacillus senioris, Lactobacillus senmaizukei, Lactobacillus sharpeae, Lactobacillus shenzhenensis, Lactobacillus sicerae, Lactobacillus silagei, Lactobacillus silagincola, Lactobacillus siliginis, Lactobacillus similis, Lactobacillus sobrius, Lactobacillus songbeiensis, Lactobacillus songhuajiangensis, Lactobacillus spicheri, Lactobacillus suantsaicola, Lactobacillus suantsaii, Lactobacillus suantsaiihabitans, Lactobacillus sucicola, Lactobacillus suebicus, Lactobacillus suibinensis, Lactobacillus sunkii, Lactobacillus suntoryeus, Lactobacillus taiwanensis, Lactobacillus tangyuanensis, Lactobacillus terrae, Lactobacillus thailandensis, Lactobacillus thermotolerans, Lactobacillus timberlakei, Lactobacillus timonensis, Lactobacillus tongjiangensis, Lactobacillus trichodes, Lactobacillus tucceti, Lactobacillus uli, Lactobacillus ultunensis, Lactobacillus uvarum, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus versmoldensis, Lactobacillus vespulae, Lactobacillus vini, Lactobacillus viridescens, Lactobacillus vitulinus, Lactobacillus wasatchensis, Lactobacillus wuchangensis, Lactobacillus xiangfangensis, Lactobacillus xujianguonis, Lactobacillus xylosus, Lactobacillus yamanashiensis, Lactobacillus yichunensis, Lactobacillus yilanensis, Lactobacillus yonginensis, Lactobacillus zeae, Lactobacillus zhachilii, Lactobacillus zhaodongensis, Lactobacillus zhaoyuanensis, Lactobacillus zhongbaensis, Lactobacillus zymae, Lactobacillus sp.

The microbial formulation according to the present invention also relates to an embodiment wherein the moisture content of the microbial formulation is between 1% and 30% and water activity is between 0.03 and 0.5. In a preferred embodiment, the moisture content of the microbial formulation is below 15% and the water activity is below 0.30.

An aspect of the invention is directed to a method of protecting the cell wall of a microorganism to dessication or drying, said method comprising the use of non-animal source protein as cryoprotectant. In an embodiment, the method comprises the further use of a disaccharide, including cellobiose, chitobiose, gentiobiose, gentiobiulose, isomaltose, kojibiose, lactose, lactulose, laminaribiose, maltose (e.g. maltose monohydrate, anhydrous maltose), maltulose, mannobiose, melibiose, melibiulose, nigerose, palatinose, rutinose, rutinulose, sophorose, sucrose, trehalose, turanose, and/or xylobiose. In a preferred embodiment, the disaccharide is maltose.

As stated, an aspect of the invention is directed to microbial formulation comprising one or more micro-organisms and a desiccation protectant comprising one or more non-animal source proteins, where said microorganism are viable after dessication such that i. at least 45% of the cells are viable after 10 weeks at 4°C, suitably under Dessication Method 1 ; and/or ii. at least 90% of the cells are viable after 6 weeks at 4°C, suitably under Dessication Method 2; and/or iv. at least 20% of the cells are viable after 4 weeks at 25°C, suitably under Dessication Method 3.

Typically, the microorganisms are viable after dessication such that i. at least 45% of the cells are viable after 10 weeks at 4°C, suitably under Dessication Method 1 ; and/or ii. at least 95% of the cells are viable after 6 weeks at 4°C, suitably under Dessication Method 2; and/or iii. at least 20% of the cells are viable after 4 weeks at 25°C, suitably under Dessication Method 3.

Particular embodiments of the present disclosure are described in the following numbered paragraphs:

1. A process of preparing a dry microbial formulation comprising: a. adding at least one desiccation protectant to a fermentate of at least one microorganism, b. mixing the at least one desiccation protectant with the fermentate to obtain a homogenous blend; and c. drying the homogenous blend, wherein the at least one desiccation protectant comprises at least one non-animal source protein and wherein the at least one desiccation protectant enables the micro- organism to have desiccation tolerance. The process of paragraph 1 , wherein the micro-organisms in the dry microbial formulation has same or higher survival rate after drying compared to the same microorganisms formulated with a desiccation protectant comprised of an animal source protein. The process of paragraph 1 or 2, wherein the non-animal source protein is a plant source protein or microbial source protein. The process of any one of paragraphs 1 to 3, wherein the plant souce protein is a protein from cereal, pseudo cereal, grain, legume, pulse, vegetable, root, nuts, hemp, chia seeds, or flax seeds. The process of any one of paragraphs 1 to 4, wherein the non-animal source protein is a peptone. The process of any one of paragraphs 1 to 5, wherein the peptone is soy peptone. The process of any one of paragraphs 1 to 3, wherein the microbial source protein is protein from yeast, yeast extract, single cell protein, or mycoproteins. The process of paragraph 1 , wherein the at least one micro-organism is lactic acid bacterium. The process of any one of paragraphs 1 to 8, wherein the lactic acid bacterium is selected from a group consisting of Pediococcus pentosaceus, Pediococcus acidilactici, Lactobacillus salivarius, Weissella confusa, Lactobacillus parafarraginis, Lactobacillus plantarum, and Lactobacillus reuteri. The process of paragraph 1 , wherein the at least one desiccation protectant is a cryoprotectant. The process of paragraph 1 , wherein the cryoprotectant is a liquid cryoprotectant. The process of any one of paragraphs 1 to 11 , wherein the liquid cryoprotectant comprises a non-animal source protein, a sugar, and water. The process of any one of paragraphs 1 to 12, wherein the sugar is maltose. The process of any one of paragraphs 1 to 13, wherein the liquid cryoprotectant comprises 1% to 30% of non-animal source protein, 0.1% to 30 % of sugar, and water. The process of any one of paragraphs 1 to 14, wherein the liquid cryoprotectant comprises 5-10% non-animal source protein, 0.1 to 3% of sugar, and water. The process of any one of paragraphs 1 to 15, wherein the liquid cryoprotectant comprises 8.85% of non-animal source protein, 2.65% of sugar, and 88.50 % of water. The process of any one of paragraphs 1 to 16, wherein the liquid cryprotectant comprises 8.85% of soytone, 2.65% of maltose, and 88.50 % of water. The process of paragraph 1 , wherein the homogenous blend is freezed before freeze- drying. Use of the dry microbial formulation of paragraph 1 in animal feed composition. Use of the dry microbial formulation of paragraph 1 as animal feed additive. A microbial formulation comprising at least one desiccation protectant and a fermentate of at least one micro-organism, wherein the at least one desiccation protectant comprises a non-animal source protein and wherein the at least one desiccation protectant enables the micro-organism to have desiccation tolerance. The microbial formulation of paragraph 21 , wherein the microbial formulation is a dry formulation. The microbial formulation of paragraph 21 , wherein the micro-organisms in the microbial formulation has same or higher survival rate after drying compared to the same micro-organisms formulated with a desiccation protectant comprised of an animal source protein. 24. The microbial formulation of paragraph 21 , wherein the at least one desiccation protectant is a cryoprotectant.

25. The microbial formulation of any one of paragraph 21 to 24, wherein the cryoprotectant is a liquid cryoprotectant when added to the fermentate and before eventually drying the microbial formulation.

26. The microbial formulation of any one of paragraphs 21 to 25, wherein the microbial formulation comprises 1.5 grams to 3.5 grams of the desiccation cryoprotectant per 1 gram of the fermentate before drying.

27. The microbial formulation of any one of paragraphs 21 to 26, wherein the moisture content is below 15% and the water activity is below 0.30.

EXAMPLES

The following examples are not intended to be a detailed catalogue of all the different ways in which the present disclosure may be implemented or of all the features that may be added to the present disclosure. Subjects skilled in the art will appreciate that numerous variations and additions to the various embodiments may be made without departing from the present disclosure. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention and not to exhaustively specify all permutations, combinations and variations thereof.

Unless otherwise indicated, the percentages set forth in the following examples are by weight, based upon the total weight of the composition.

EXAMPLE 1

Preparation of a desiccation protectant containing non-animal source protein

Dry components i.e. soy peptone and maltose were weighed out as per the weight percent mentioned in the Table 1. The dry components were added to a large beaker or carboy type vessel. Water was added to the dry components to form a desiccation protectant solution. The desiccation protectant solution was mixed until the dry component were dissolved in water. The desiccation protectant solution was autoclaved at 121°C for 15 minutes to 30 minutes. The desiccation protectant solution was stored at room temperature until time of use.

Table 1.

EXAMPLE 2

Process of preparing a freeze-dried formulation of lactic acid bacteria

Figure 1 illustrates a flow diagram for a process of preparing a freeze-dried formulation of lactic acid bacteria

Fermentate:

A fermentate was used as a starting material. The fermentate was prepared by a liquid fermentation of a lactic acid bacteria.

Recovery:

In the process of recovery, the liquid fermentate was concentrated to paste using a decanter style centrifuge. The resulting paste was typically 25-40% solids.

Preparation of the formulation:

The paste was added to a kitchen blender and the liquid cryoprotectant prepared in Example 1 was added to the kitchen blender with the paste at a ratio of 1.5-3.5 grams of liquid cryoprotectant per 1 gram of paste. The paste and liquid cryoprotectant was blended for 1 to 2 minutes, until a homogeneous blend was formed.

Freezing:

The homogenous blend was transferred to freeze drying trays or similar vessels. The homogenous blend had a bed thickness in the tray of 0-1 inches. The homogenous blend in trays were either snap frozen by pouring liquid nitrogen on the solution or by storing at -80°C until frozen (typically overnight).

Freeze Drying:

The frozen trays were transferred to a freeze dryer. The samples were freeze-dried for approximately 24 hours or until dried. The freeze-dried sample had a moisture content below 15% and a water activity below 0.30.

Further Processing:

The freeze-dried sample was either put into storage or blended into other products as the active ingredient.

EXAMPLE 3

Microbial Stability of freeze-dried lactic acid bacteria Pediococcus acidilactici and Wiesella confusa using a non-animal source protein as a desiccation protectant

Lactic acid bacteria strains Pediococcus acidilactici FM18 (deposited as NRRL Deposit Number B-50964) and Wiesella confusa (deposited as Wiesella cibaria NRRL B-50961 in 2014 and updated as Wiesella confusa in 2016) were processed separately using identical workflows as described in Example 2. Each strain was harvested post fermentation and concentrated down to a paste via centrifugation (15,000 rpm for 20 min) in 1 L bottles. The resultant paste of each strain was transferred to a blender where the paste was combined with cryoprotectants as shown in Table 2 mixing until a homogenous blend is obtained.

Table 2.

An aliquot of the homogenous blend of each sample was poured into freeze drying trays to a bed thickness of 0.5 inches and frozen at -80°C for 24 hours. Each of the frozen homogenous sample was freeze-dried in a Millrock Revo Freeze Dryer (Millrock Technology, Kingston, NY) for approximately 24 hours. The resultant powders of each sample were packaged, and vacuum sealed in plastic bags. The vacuum sealed plastic bags of the samples were stored in a refrigerator at 4°C. The samples were assayed for viability over the course of 10 weeks as shown in Figure 2.

(Dessication Method 1)

Table 3. The results in Figure 2 and Table 3 demonstrates that the non-animal source protein used as a desiccation protectant provides improved or equal microbial stability to the lactic acid bacteria as provided by the animal source protein.

EXAMPLE 4

Microbial stability of spray-dried lactic acid bacteria Wiesella confusa using a non-animal source protein as a desiccation protectant

Lactic acid bacteria strain Wiesella confusa was harvested post fermentation and concentrated down to a paste via centrifugation (15,000 rpm for 20 min) in 1 L bottles. The resultant paste was transferred to a blender where the paste was combined with desiccation protectants as shown in Table 4 by mixing until a homogenous blend is obtained.

Table 4.

An aliquot of the homogenous blend of each sample was spray-dried using a BUCHI Mini Spray Dryer B-290 (BUCHI Corp., New Castle, DE) equipped with a two-fluid nozzle. The flow rate of the fluid was 7.5 mL/min. The inlet temperature was 122-135°C and the outlet temperature was controlled to 65°C. The aspirator and regulator were both run at the maximum setting using room air at ambient temperature and humidity. The resultant powders were packaged, and vacuum sealed in plastic bags. The vacuum sealed plastic bags of the samples were stored in refrigerator at 4°C. The samples were assayed for survivability over the course of 6 weeks as shown in Figure 3 and Table 5. (Dessication Method 2)

Table 5 The results in Figure 3 and Table 5 demonstrates that the non-animal source protein used as a desiccation protectant provides improved microbial stability to the lactic acid bacteria as provided by the animal source protein.

EXAMPLE 5

Microbial stability of freeze-dried Lactobacillus reuter using a soytone containing cryoprotectant

Lactic acid bacteria strain Lactobacillus reuteri (deposited as DSM 17648) was harvested post fermentation and concentrated down to a paste via centrifugation (15,000 rpm for 20 min) in 1 L bottles. The resultant paste was transferred to a blender where the paste was combined with cryoprotectant as shown in Table 6, mixing until a homogenous blend was obtained.

Table 6.

An aliquot of Sample 1 was poured into a freeze-drying tray to a bed thickness of 0.5 inches and frozen at -80°C for 24 hours. The frozen homogenous sample was freeze-dried in a Millrock Revo Freeze Dryer (Millrock Technology, Kingston, NY) for approximately 24 hours. The resultant powder was analyzed for microbial recovery after drying. The remaining powder was packaged, and vacuum sealed in mylar bags. The vacuum sealed mylar bags of the samples were stored in a refrigerator at 25°C. The samples were assayed for viability to assess their microbial stability after 4 weeks of storage.

Table 7.

The results in Table 7 demonstrates that the non-animal source protein used as a desiccation protectant provides sufficient desiccation protections during freeze drying to the Lactobacillus reuteri as evident by the percent microbial recovery after drying. (Dessication Method 3)